[Protein-analysis] PEGylated peptide separation via CEX

boyu du via proteins%40net.bio.net (by du.boyu from gmail.com)
Wed Mar 2 03:37:33 EST 2011

Dear Mailing list:

Now I am trying to PEGylated a small peptide (MW is about 4KD with pI value
as 4.5). The PEG molecule I used was a bifunctional PEG molecule. I just
conjugated one of these two functional ends to the amino group of the
peptide and left the other active for further experimental process. Source S
resin was adopted to separate the PEGylated form from un-PEGylated form as
well as different isomers. But unfortunately, I observed that all of these
different forms were hard to be eluted by the buffer I selected.

Both pH 2 and pH 3 buffer system were tested. Isopropanol, ethanol, ACN,
Urea, ethylene glycol were all tested  to combine into the buffer to elute
the PEGylated and un-PEGylated form of peptide. When ethanol and ACN was
included in the buffer at pH2, the un-PEGylated peptide could be eluted. But
the PEGylated form could not.

I have tried many times with the above conditions to elute the PEGylated
form and tried to separate the different isomer via a gradient elution mode.
But all these trials were failed.

RP-HPLC has also tested to separate my target peptide. But it had been ruled
out as it could not separate the isomers formed during the PEGylation
process and the lyophilization process could inactivate the other active
function end of PEG molecule.

What should I do to solve this problem? Is anyone here has the experiences
to deal with small PEGylated peptide purification?

Thank you all in advance!


Boyu Du

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